【摘要】 本研究以8周龄的吉林白鹅胸肌组织为材料,利用SMART技术构建全长cDNA文库的方法,进行大片段双链cDNA扩增。经内切酶SfiⅠ酶切并以改造后具有SfiⅠA和SfiⅠB接头序列的pB luescriptⅡSK质粒为连接载体,进行cDNA克隆,构建质粒cDNA文库。结果表明,所构建的肌肉组织cDNA文库滴度为1.01×106pfu/mL,质粒的转化重组率为97%,插入cDNA片段的大小在0.25~1.6 kb之间。随机取24个重组克隆子进行测序,所获得的un igene比率为66.7%,全长cDNA序列完整性比率为80%。将全长cDNA序列与GenBank数据库进行核苷酸同源性比较,发现其中一cDNA序列为鹅泛素Ⅰ基因。该基因的编码序列长918 bp,编码由305个氨基酸组成的多聚泛素蛋白。鹅泛素ⅠcDNA序列编码区与鸡等16种动物的同源序列比较显示,其相似性为87.3%~93.7%。该文库的构建为在分子水平上研究鹅肌肉组织的生长发育奠定了基础。更多还原

【Abstract】 The muscular tissue of breast was dissected from Jilin White goose at 8 weeks of age in the present study.The long-distance PCR method was used to amplify double-strand cDNA based on the SMART techniques for construction of a full-length cDNA library.After digestion with restriction endonuclease SfiⅠ,a modified vector of pBluescript II SK plasmid with the adaptors containing SfiⅠSfiⅠA and SfiⅠB sites was used to recombine with the cDNA products amplified.The recombinants were cloned by transformation into competent Escherichia coli DH2α.A plasmid cDNA library with goose muscle was constructed.The results showed that the titer of the cDNA library is 1.01×106 pfu /ml,in which the percentage of recombinant clones is 97%.The length of most cDNA inserted was between 0.25 kb and 1.1 kb identified by gel electrophoresis after cDNA PCR amplification.The unigene ratio was 66.7% and the percentage of complete cDNA sequences was 80% by estimating from the 24 clones sequenced randomly.A full-length anUbⅠcDNA was found by aligned with those of its homologues in the NCBI database using the BlastN program,and the accession number in GenBank is DQ420616.The sequence length of coding region of anUbⅠcDNA is 918 bp,it encodes 305 amino acids consisting of the polyubiquitin protein.By comparison,high similarities of 87.3-93.7% in the coding region sequences of polyubuquitin genes were found in 16 species including chicken.It is helpful to study muscle development of goose at molecular level in the future.更多还原